1. Field of the Invention
The invention relates to embryoid body systems and methods employing embryoid bodies for the screening of chemical and biological agents for safety and effectiveness.
2. Description of the Related Art
Embryonic stem (ES) cells are pluripotent cells derived from an early embryonic stage of development. When cultured in the presence of leukocyte inhibitory factor (LIF), ES cells remain undifferentiated. Removal of LIF and regulation of culture conditions lead to ES cell differentiation into specific cell types [1-7]. Many differentiation strategies involve the generation of embryoid bodies (EB) that are aggregates of ES cells in suspension [8]. The addition of small molecules to EB has three potential outcomes: 1) increased differentiation of EB into particular lineages or cell types; 2) toxic effects on the EB; or 3) inhibition of particular lineages or cell types. As an example, the presence of the small molecule retinoic acid (RA) at an appropriate time point is one strategy for differentiation of ES cells into neuronal cell types. An object of this invention is that embryoid bodies (which contain all germ layers: ectoderm, mesoderm, endoderm) are useful for the study of small molecule effects on differentiation of ES cells into cell types of each germ layer.
ES cells are totipotent and have been used as model systems for differentiation studies. ES cells are derived from the inner cell mass of developing blastocysts, are maintained in culture under appropriate conditions (such as growth on embryonic fibroblasts in the presence of LIF) where they retain their totipotency and are able to generate all cell lineages after introduction into host blastocysts [9-11]. Using appropriate culture conditions, ES cells differentiate and form EBs that contain cells of hematopoietic, endothelial, muscular and neuronal lineages [6, 7, 12, 13]. Many aspects of lineage-specific differentiation programs observed within EBs reflect those found in the embryo, indicating that this model system provides access to early cell populations that develop in a normal fashion. ES cells are also able to spontaneously differentiate and generate various lineages under appropriate conditions in cell culture [6, 7, 12, 13]. The in vitro differentiation potential of ES cells allows their use as a model system for the study of developmental potential and also as a valuable reagent for stem cell therapeutic approaches. The instant invention disclosed herein is directed to an efficient in vitro system to screen and study effects of small molecules and bioagents, and is an alternative to embryo and live animal studies. Using this in vitro system, it is possible to screen many compounds efficiently and, once a candidate molecule is identified, this agent can be studied thoroughly to define how it exerts its effect.